Extract with anti-tumor and anti-poisonous activity

ABSTRACT

Extracts of the plant  Calotropis procera , having a pharmacological activity, in particular an anti-poisonous activity, and active compounds isolated thereof are described. Furthermore, methods for the extraction of the extracts are disclosed. Also, a pharmaceutical composition or product for the treatment of cancer which includes an effective amount of the described extracts or an active compound thereof, a therapeutic compound and optionally a pharmaceutical acceptable carrier are described.

FIELD OF THE INVENTION

The present invention relates to the medical field. The inventionrelates in a first aspect to extracts of the plant Calotropis procera,having a pharmacological activity, in particular an anti-tumor activityand/or anti-poisonous activity and active compounds isolated thereof. Ina second aspect, the present invention relates to methods for obtainingsaid extracts. The invention further relates in a third aspect to apharmaceutical composition for the treatment of cancer comprising aneffective amount of said extracts or an active compound thereof. In afourth aspect, the present invention concerns the use of said extractsor an active compound thereof as a medicament and the use of saidextracts or an active compound thereof for the preparation of amedicament for the treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer develops in a given tissue when some genomic mutation perturbscell cycle kinetics by increasing cell proliferation or decreasing celldeath, or both. This perturbation leads to unrestrained growth of agenomically transformed cell population. Some cells from thistransformed cell population may switch to the angiogenic phenotype,enabling them to recruit endothelial cells from the healthy tissue andleading to the sustained growth of the developing neoplastic tumortissue. Subsequently, some cells migrate from the neoplastic tumortissue and colonize new tissues, using blood or lymphatic vessels asmajor routes of migration. This process is also known as the metastaticprocess.

In practice, most of the agents used today in hospitals to treat cancerpatients are drugs, which more or less directly target the cellkinetics, i.e. cell proliferation, of the cancer to be combated. Theworking mechanism of such anti-cancer drugs essentially relates to thedisruption of the development of malignant cells by acting on cellkinetics. These drugs include alkylating agents, intercalating agents,antimetabolites, etc., most of which target DNA or enzymes regulatingthe DNA duplication and elongation process. These drugs attack DNA.

A major drawback of these drugs involves that the drugs do not work in aselective manner, i.e. they do not select between normal and neoplasticcells. They are used in accordance with the fact that the DNA of rapidlyproliferating cells, i.e. cancer cells, is more sensitive to this typeof agents than the DNA of less rapidly proliferating cells, i.e. normalcells. However, rapidly growing tumors are not always tumors exhibitinghigh levels of cell proliferation. Rapidly growing tumors may alsoinclude tumors which exhibit low levels of cell death compared to thenormal cell population from which these tumor cells issue. For thesetypes of rapidly growing tumors, the mentioned drugs are not effective.

In addition, the great majority of the drugs used in the standardtreatment of cancer using the cell kinetics approach have the drawbackof being toxic or even highly toxic, i.e. involving many detrimentalside-effects on healthy cells, tissues and organs, and this limits theirclinical use to a relatively low number of administrations per patient.In addition, several of these compounds must be combined into apoly-chemotherapeutic regimen in order to have any observable effectagainst cancer. By way of evidence such anti-cancer drug combinationsincrease detrimentally the toxicity of the treatment and also limit thenumber of administrations that can be applied.

Some anti-cancer drugs from natural origins, such as e.g. anti-tubulincompounds, using a therapeutic approach different from the cell kineticsapproach, have been proposed. Said drugs aim to prevent the migration ofcancer cells which escape from the tumor bulk and first invadeneighboring tissue therefore establishing metastases. However, thecompounds of this type known so far also show major toxic side-effects,which limits their use over long periods of treatment.

Therefore, there remains an urgent need in the art for finding improvedanti-cancer drugs, which overcome at least some of the above-mentioneddrawbacks. Consequently, it is a general object of the invention toprovide improved anti-cancer drugs. In particular the invention aims toprovide an improved anti-cancer drug, showing minimal side effects.

SUMMARY OF THE INVENTION

One embodiment of the present invention is an extract of the plantCalotropis procera, characterized in that said extract has apharmacological activity, in particular an anti-poisonous activity.

Another embodiment of the present invention is an extract as describedabove obtained using an extraction procedure, comprising the steps of:

-   -   a) extracting the starting material of said Calotropis procera        plant, said starting material being selected among fruits,        aerial parts subterranean parts, and their mixtures, in an        aliphatic alcohol, by dissolving the starting material in said        alcohol thereby obtaining a suspension of said material in said        alcohol, stirring said suspension, and filtering said suspension        by fritted glass thereby obtaining a first filtrate and a first        solid part;    -   b) extracting said first solid part in an aliphatic alcohol        thereby obtaining a second filtrate and a second solid part;    -   c) combining said first and said second filtrate thereby        obtaining a combined filtrate, and evaporating said combined        filtrate under vacuum thereby obtaining said extract.

Another embodiment of the present invention is an extract as describedabove obtained using a extraction procedure comprising the steps:

-   -   a) grinding the starting material of leaf blades, stems, barks        and roots of Calotropis procera to give a fine powder of the        plant,    -   b) extracting the powder of step a) with dichloromethane for at        least 6, 12, 18 or preferably 24 hours using a soxhlet        extractor,    -   c) decanting the dichloromethane of step b), and evaporating the        filtrate, after filtration, to obtain a gum.

Another embodiment of the present invention is an extract as describedabove obtained using a extraction procedure comprising the steps:

-   -   a) grinding the starting material of leaf blades, stems, barks        and roots of Calotropis procera give a fine powder of the plant,    -   b) extracting the powder of step a) with dichloromethane for at        least 6, 12, 18 or preferably 24 hours using a soxhlet        extractor,    -   c) decanting the dichloromethane of step b), and evaporating the        filtrate, after filtration, to obtain a gum,    -   d) extracting the residue of step c) with methanol for at least        6, 12, 18 or preferably 24 hours using a soxhlet extractor,    -   e) decanting the methanol of step d), evaporating the filtrate,        after filtration, to obtain a gum,    -   f) subjecting the gum of step e) to column chromatography using        flash silica gel and dichloromethane-methanol as solvent, and    -   g) collecting a first fraction and evaporating of the said        fraction to obtain a gum having biologically active components.

Another embodiment of the present invention is an extract as describedabove obtained using a extraction procedure comprising the steps:

-   -   a) grinding the starting material of leaf blades, stems, barks        and roots of Calotropis procera give a fine powder of the plant,    -   b) extracting the powder of step a) with dichloromethane for at        least 6, 12, 18 or preferably 24 hours using a soxhlet        extractor,    -   c) decanting the dichloromethane of step b), and evaporating the        filtrate, after filtration, to obtain a gum,    -   d) extracting the residue of step c) with methanol for at least        6, 12, 18 or preferably 24 hours using a soxhlet extractor,    -   e) decanting the methanol of step d), evaporating the filtrate,        after filtration, to obtain a gum,    -   f) subjecting the gum of step e) to column chromatography using        flash silica gel and dichloromethane-methanol as solvent,    -   g) collecting a first fraction, having biologically active        components,    -   h) applying the concentrated fraction of step g) to column        chromatography using flash silica gel and hexane-acetone as        solvent to give two fractions, and    -   i) washing the column after step h) with methanol to give a        third fraction, having biologically active components.

Another embodiment of the present invention is a composition comprising:

-   -   an extract of Calotropis procera as described above, and    -   at least one therapeutic compound and/or a physical treatment        that exerts relevant, detrimental side effects on normal,        non-cancer related cells, tissues or organs.

Another embodiment of the present invention is a product containing

-   -   an extract of Calotropis procera, as described above, and    -   at least one therapeutic compound and/or a physical treatment        that exerts relevant, detrimental side effects on normal,        non-cancer related cells, tissues or organs as a combined        preparation for simultaneous, separate or sequential        administration to a subject.

Another embodiment of the present invention is a composition asdescribed above or a product as describe above where one of the saidextracts comprises at least two active compounds selected from the groupcomprising asclepin, calactin, vorusharin, calotropin, calotropagenin,uzarigenin, calotoxin, usharin and usharidin.

Another embodiment of the present invention is a composition asdescribed above, or a product as describe above wherein one of the saidextracts comprises at least one of the compounds which are representedin Table 1.

Another embodiment of the present invention is a composition asdescribed above or a product as describe above wherein the weight ratioof extract: therapeutic compound is in the range 0.001:1 to 1000:1.

Another embodiment of the present invention is a composition asdescribed above, or a product as describe above, for use as amedicament.

Another embodiment of the present invention is a composition asdescribed above, or a product as describe above, for use as a medicamentfor the treatment of cancer.

Another embodiment of the present invention is a composition or productas described above, wherein said cancer is selected from the groupcomprising breast cancer, lymphoma, sarcoma, pancreatic cancer,melanoma, colorectal cancer, glioma, non small cell lung cancer, smallcell lung cancer, skin cancer, bone cancer, ovarian cancer, CNS cancer,renal cancer, bladder cancer, head and neck cancer, prostate cancer,liver cancer, hematological cancers.

Another embodiment of the present invention is a composition asdescribed above or a product as describe above further comprising one ormore additional therapeutic compounds.

Another embodiment of the present invention is a composition asdescribed above, or a product as describe above, wherein saidtherapeutic compound(s) is an anti-cancer agent.

Another embodiment of the present invention is a composition asdescribed above, or a product as describe above, wherein saidtherapeutic compound is selected from the group comprising adriamycin,alkeran, ara-c, bleomycin, biCNU, busulfan, CCNU, carboplatinum,cisplatinum, cyclophosphamide, cytoxan, daunorubicin, DTIC, 5-FU,fludarabine, gemcitabine (gemzar), herceptin, hexamethylmelamine,hydrea, idarubicin, ifosfamide, irinotecan (camptosar, CPT-11),leustatin, methotrexate, mithramycin, mitomycin, mitoxantrone, muphoran,navelbine, nitrogen mustard, oxaliplatine, rituxan, STI-571,streptozocine, taxol, taxotere, topotecan (hycamtin), velban,vincristine, VP-16, xeloda (capecitabine), or zevelin.

Another embodiment of the present invention is a composition asdescribed above, or a product as describe above, wherein saidtherapeutic compound is selected from the group comprising adriamycine,vincristine, camptothecin and oxaliplatin.

Another embodiment of the present invention is a composition asdescribed above, or a product as describe above, wherein saidtherapeutic compound(s) is a cytotoxic antibody or a fragment thereof.

Another embodiment of the present invention is a composition asdescribed above, or a product as describe above, wherein saidtherapeutic compound(s) is a cytotoxic hormone or a fragment thereof.

Another embodiment of the present invention is a composition asdescribed above, or a product as describe above, wherein saidtherapeutic compound(s) is a cytotoxic peptide or a fragment thereof.

Another embodiment of the present invention is a composition asdescribed above further comprising a pharmaceutically acceptable carrieror a product as describe above wherein the composition and/ortherapeutic compound(s) further comprises a pharmaceutically acceptablecarrier.

Another embodiment of the present invention is a use of an extract ofCalotropis procera as described above for the preparation of amedicament for alleviating the side effects of one or more therapeuticcompounds.

Another embodiment of the present invention is a use of an extract ofCalotropis procera as described above for the preparation of amedicament for increasing the dose administered to an individual of oneor more therapeutic compounds.

Another embodiment of the present invention is a use of an extract asdescribed above wherein said therapeutic compound(s) has anti-canceractivity.

Another embodiment of the present invention is a use of an extract asdescribed above wherein said therapeutic compound(s) is selected fromthe group comprising adriamycin, alkeran, ara-c, bleomycin, biCNU,busulfan, CCNU, carboplatinum, cisplatinum, cyclophosphamide, cytoxan,daunorubicin, DTIC, 5-FU, fludarabine, gemcitabine (gemzar), herceptin,hexamethylmelamine, hydrea, idarubicin, ifosfamide, irinotecan(camptosar, CPT-11), leustatin, methotrexate, mithramycin, mitomycin,mitoxantrone, muphoran, navelbine, nitrogen mustard, oxaliplatine,rituxan, STI-571, streptozocine, taxol, taxotere, topotecan (hycamtin),velban, vincristine, VP-16, xeloda (capecitabine), or zevelin.

Another embodiment of the present invention is a use of an extract asdescribed above wherein said therapeutic compound(s) is selected fromthe group comprising adriamycine, vincristine, camptothecin andoxaliplatin.

Another embodiment of the present invention is a use of an extract asdescribed above wherein said therapeutic compound(s) is a cytotoxicantibody or a fragment thereof.

Another embodiment of the present invention is a use of an extract asdescribed above wherein said therapeutic compound(s) is a cytotoxichormone or a fragment thereof.

Another embodiment of the present invention is a use of an extract asdescribed above wherein said therapeutic compound(s) is a cytotoxicpeptide or a fragment thereof.

Another embodiment of the present invention is a use of an extract asdescribed above wherein said therapeutic compound(s) is therapeuticradiation.

Another embodiment of the present invention is a use of an extract asdescribed above wherein said extract comprises at least two activecompounds selected from the group comprising asclepin, calactin,vorusharin, calotropin, calotropagenin, uzarigenin, calotoxin, usharinand usharidin.

Another embodiment of the present invention is a use of an extract asdescribed above wherein said extract further contains at least one ofthe compounds which are represented in Table 1.

Another embodiment of the present invention is a use of an extract asdescribed above wherein said extract is administered prior to saidtherapeutic compound(s).

Another embodiment of the present invention is a use of an extract asdescribed above wherein said extract is administered after saidtherapeutic compound(s).

Another embodiment of the present invention is a use of an extract asdescribed above wherein said extract is administered at the same time assaid therapeutic compound(s).

Another embodiment of the present invention is a use of an extract asdescribed above wherein the weight ratio of extract:therapeutic compoundis in the range 0.001:1 to 1000:1.

Another embodiment of the present invention is an extraction process forobtaining an extract having biologically active components comprisingthe steps of:

-   -   a) extracting the starting material of said Calotropis procera        plant, said starting material being selected among fruits,        aerial parts, subterranean parts, and their mixtures, in an        aliphatic alcohol, by dissolving the starting material in said        alcohol thereby obtaining a suspension of said material in said        alcohol, stirring said suspension; and filtering said suspension        by fritted glass thereby obtaining a first filtrate and a first        solid part;    -   b) extracting said first solid part in an aliphatic alcohol        thereby obtaining a second filtrate and a second solid part;    -   c) combining said first and said second filtrate thereby        obtaining a combined filtrate; and    -   d) evaporating said combined filtrate under vacuum thereby        obtaining said extract.

Another embodiment of the present invention is an extraction process forobtaining a several extracts having biologically active componentspresent substantially in the leaves, stems, barks and roots ofCalotropis procera, which comprises the following steps:

-   -   a) grinding the starting material of leaf blades, stems, barks        and roots of Calotropis procera give a fine powder of the plant,    -   b) extracting the powder of step a) with dichloromethane for at        least 6, 12, 18 or preferably 24 hours using a soxhlet        extractor, and    -   c) decanting the dichloromethane of step b), and evaporating the        filtrate, after filtration, to obtain a gum.

Another embodiment of the present invention is an extraction process asdescribed above further comprising the steps of:

-   -   d) extracting the residue of step c) with methanol for at least        6, 12, 18 or preferably 24 hours using a soxhlet extractor,    -   e) decanting the methanol of step d), evaporating the filtrate,        after filtration, to obtain a gum,    -   f) subjecting the gum of step e) to column chromatography using        flash silica gel and dichloromethane-methanol as solvent, and    -   g) collecting a first fraction having biologically active        components, and evaporating the eluent to obtain said extract.

Another embodiment of the present invention is an extraction process asdescribed above, further comprising the steps of

-   -   h) applying fraction of step g) to column chromatography using        flash silica gel and hexane-acetone as solvent to give two        fractions,    -   i) washing the column after step h) with methanol to give a        third fraction, having biologically active components

Another embodiment of the present invention is a process as describedabove, wherein step b) is performed at a working temperature of between20 and 80° C.

Another embodiment of the present invention is a process as describedabove, wherein said step b) is repeated between one and five times.

Another embodiment of the present invention is a process as describedabove, wherein the duration of step b) is between 4 hours and 48 hours.

Another embodiment of the present invention is a process as describedabove, wherein the solid phase of step d) is silica gel.

Another embodiment of the present invention is a process as describedabove, wherein the eluent of step d) is a binary eluent, the ratiobetween the two components of the eluent being between 100:0 to 0:100.

Another embodiment of the present invention is a process as describedabove, wherein the components of said binary eluent comprise analcoholic solvent and a non polar solvent.

Another embodiment of the present invention is a process as describedabove, wherein step e) is performed at a working temperature of between20 and 50° C.

Another embodiment of the present invention is a process as describedabove, wherein the solid phase of step f) is silica gel.

Another embodiment of the present invention is a process as describedabove, wherein the eluent of step f) is a binary eluent, the ratiobetween the two components of eluent being between 100:0 to 0:100.

Another embodiment of the present invention is a process as describedabove, wherein the components of the binary eluent are a non-polar and amore polar solvent.

Another embodiment of the present invention is a process as describedabove, wherein the ratio between the two components of eluent,non-polar:polar, is between 50:50 and 100:0.

Another embodiment of the present invention is a process as describedabove, wherein the solvent of step h) is methanol.

Another embodiment of the present invention is a process as describedabove, wherein step e) is performed at a working temperature of between20 and 50° C.

Another embodiment of the present invention is an active extractisolated from the process as described above.

Another embodiment of the present invention is a use of an activeextract as described above as a medicament.

Another embodiment of the present invention is a use of an activeextract as described above for the preparation of a medicament in thetreatment of cancer.

Another embodiment of the present invention is a method for treatingcancer comprising administering to an individual in need of suchtreatment a pharmaceutical composition as described above, or a productas described above.

Another embodiment of the present invention is a method as describedabove, wherein said cancer is selected from the group comprising breastcancer, lymphoma, sarcoma, pancreatic cancer, melanoma, colorectalcancer, glioma, non small cell lung cancer, small cell lung cancer, skincancer, bone cancer, ovarian cancer, CNS cancer, renal cancer, bladdercancer, head and neck cancer, prostate cancer, liver cancer,hematological cancers.

Another embodiment of the present invention is a composition asdescribed above, or product as described above, wherein the therapeuticcompound is radiation.

Another embodiment of the present invention is a kit comprising acontainer in which an extract of Calotropis procera as described aboveis present, and a container in which a therapeutic compound is present.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to extracts of the plant Calotropisprocera. In a first embodiment the invention relates to extracts of theplant Calotropis procera, which has a pharmacological activity, inparticular an anti-tumor activity and/or an anti-poisonous activity.Surprisingly, at least one of the extracts according to the inventioncombines an anti-tumor effect with an anti-poisonous activity.

According to the present invention the term “anti-tumor activity”,refers to the in vitro as well as in vivo anti-tumor effects exerted byat least one of the extracts or isolated compounds thereof. Theanti-tumor effects essentially include but are not limited to a dramaticdecrease of cell growth and a pro-apoptotic effect. Importantly, theextracts according to the invention exhibits anti-tumor activity on alarge number of cancer types, such as breast cancer, melanoma orlymphoma cancers amongst others.

Another feature of the extracts according to the invention encompassestheir anti-poisonous activity. The term “anti-poisonous activity” refersto the ability of the extracts according to the invention or isolatedcompounds thereof to attenuate or reverse the effects of therapeuticcompounds or treatments. A “therapeutic compound”, as used herein,refers to a compound that exerts a relevant detrimental, toxic effect(s)on normal, i.e. non-cancer related cells, tissues or organs. The termtherapeutic compound may thus also include a compound, drug, physicaltreatment or medicament, used to treat a particular disease that exertsdetrimental toxic side effects. Such side effects are known to theskilled person and include, but are not limited to tiredness, nausea,vomiting, sores, mouth sores, dry skin, sensitive skin, hair loss,reduced red and white blood count. Such therapeutic compounds include,but are not limited to chemotherapy agents, radiation, antibodies andhormones.

Therapeutic compounds useful according to the invention include thoseused in chemotherapy or are cytotoxic. They include, but are not limitedto any of adriamycin, alkeran, ara-c, bleomycin, biCNU, busulfan, CCNU,carboplatinum, cisplatinum, cyclophosphamide, cytoxan, daunorubicin,DTIC, 5-FU, fludarabine, gemcitabine (gemzar), herceptin,hexamethylmelamine, hydrea, idarubicin, ifosfamide, irinotecan(camptosar, CPT-11), leustatin, methotrexate, mithramycin, mitomycin,mitoxantrone, muphoran, navelbine, nitrogen mustard, oxaliplatine,rituxan, STI-571, streptozocine, taxol, taxotere, topotecan (hycamtin),velban, vincristine, VP-16, xeloda (capecitabine), or zevelin.

It is another aspect of the invention that a therapeutic agent comprisesone or more anticancer agents which are derived from an extract of thepresent invention. Possible anti-cancerous compounds according to theinvention are any extracted from Calotropis procera such as asclepin,calactin, voruscharin, calotropin, calotropagenin, uzarigenin,calotoxin, uscharin and uscharidin.

In another embodiment of the invention, the anti-cancerous compounds areany extracted from Calotropis procera such as 2″oxo-voruscharin, andderivatives thereof as shown in Table 2.

It is another aspect of the invention that a therapeutic agent is anextract of the invention. Said extract may be identical to an extractthat has an anti-poisonous activity. Alternatively, said extract may beidentical to an extract that has an anti-poisonous activity, with theexception of the absence of one or more active compounds from eitherextract. Alternatively, said extract may be identical to an extract thathas an anti-poisonous activity, with the exception of the presence ofone or more additional active compounds in either extract.Alternatively, said extract may be identical to an extract that has ananti-poisonous activity, with the exception of difference in theconcentration of one or more active compounds in either extract.

It is another aspect of the invention that a therapeutic compound is aphysical treatment such as radiation. Said radiation may be any used inmedicine. For example the radiation may be that used to shrink tumourousgrowths, to totally irradiate the body as part of a bone marrowtransplants program, etc. The radiation may also be that used fordiagnosis, for example, in X-rays or radioactive markers.

It is another aspect of the invention that the therapeutic compound isan antibody, or a fragment thereof. Said antibody demonstrates cytotoxicactivity. Examples of therapeutic antibodies include, but are notlimited to HerceptinR, Prostascint, Rituximab, and Trastuzumab.

It is another aspect of the invention that the therapeutic compound is ahormone, or a fragment thereof. Said hormone demonstrates cytotoxicactivity. Examples of therapeutic hormones include, but are not limitedto busereline, fluoxymesterone, flutamide, formestane, norethandrolone,norethisterone, prednisolone, prednisone, and tamoxifene

It is another aspect of the invention that the therapeutic compound is apeptide, or a fragment thereof. Said peptide demonstrates cytotoxicactivity.

By fragment in reference to a antibody, hormone or peptide means apeptide comprising a portion of the antibody, hormone or peptide that iscapable of recognizing the target of the whole antibody, hormone orpeptide. The fragment is also capable of recognizing the target of thewhole antibody, hormone or peptide. The portion may comprise amino acidsubstitutions, deletions or insertions that do not substantially changethe recognition of the target by portion. The number of substitutions,deletions or insertions may be less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 200, 300, 400, or 500.

It is another aspect of the invention, that the anti-poisionous activityof the extracts allows the administration of a higher dose of thetherapeutic compound. By administering a higher dose of therapeuticcompound, the cancer may be more quickly and effectively treated. Anindividual in receipt of a higher dose, in combination with an extractof the invention would benefit from reduced side-effects of thetherapeutic compound.

It is another aspect of the invention that the anti-poisionous activityof the extracts allows the administration of a combination oftherapeutic compounds. A combination therapy would target differentaspects of the cancer simultaneously, so leading to a more effective andefficient treatment. By administering the combination in accordance withthe invention, the patent would suffer less side effects and benefitfrom the effectiveness of the combination therapy.

In another embodiment, the present invention relates to the activecompounds, isolated from the extracts. The term ‘active compounds’ or‘active components’ of the extracts are used herein as synonyms, andrefer to the compounds present in the extracts, which exhibit anactivity that is similar to at least one of the above-defined activitiesof the extracts.

In yet another embodiment, the present invention relates to methods forobtaining the extracts of the plant Calotropis procera.

Due to the fact that at least one of the extracts according to theinvention exert an anti-tumor activity the extracts according to theinvention are particularly useful for the treatment of diseases such ascancer. Therefore, in another aspect, the present invention relates topharmaceutical compositions comprising one of the above-describedextracts or at least one active compound thereof.

Furthermore, since the extracts according to the invention exert ananti-poisonous activity as well, it is also particularly suitable to becombined with other therapeutic compounds, such as other medicaments,which show toxic side effects. Therefore, in another embodiment, thepresent invention relates to a pharmaceutical composition comprising oneof the above-described extracts or at least one active compound thereof,and a second compound, which exerts a pharmacological activity havingtoxic side effects.

Furthermore, the present invention relates to the use of one of saidextracts and/or at least one active compound thereof as a medicament.The present invention further relates to the use of one of the extractsor at least one active compound thereof in the preparation of amedicament for the treatment of diseases, in particular cancer.

In another embodiment, the present invention further relates to a methodfor treating cancer.

Other objects and advantages of the present invention will becomeapparent from the following detailed description taken in conjunctionwith the accompanying drawings.

FIGURES

FIG. 1 describes the overall growth of different types of tumors as afunction of the concentration of an extract according to the invention.

FIGS. 2 to 11 show the in vitro effects of an extract according to theinvention on different types of human cancer cell lines, being breastcancer cell lines in FIG. 2, sarcoma cancer cell lines in FIG. 3,pancreatic cancer cell lines in FIG. 4, melanoma cancer cell lines inFIG. 5, colon cancer) cell lines in FIG. 6, glioma cancer cell lines inFIG. 7, lung cancer cell lines in FIG. 8, bladder cancer cell lines inFIG. 9, prostate cancer cell lines in FIG. 10, and head and neck cancercell lines in FIG. 11.

FIGS. 12 to 16 represent the in vitro effects of an extract according tothe invention on the cell cycle kinetics of cells corresponding to fivedifferent human cell lines, HCT-15 (FIG. 12), RPMI (FIG. 13), A172 (FIG.14), J82 (FIG. 15) and Hs683 (FIG. 16). The upper part of the figuresshows overall cell growth, as the percentage of living cancer cells tocontrol cells, at different doses of the extract. The middle and lowerparts of the figures show the effect on the cell cycle kinetics of thesaid extract at different concentrations after 24 hours and 72 hoursrespectively. Statistical significance was evaluated with the Student'st-test where *: p<0.05; **: p<0.01: ***: p<0.001.

FIGS. 17 to 20 represent the in vitro apoptotic-inducing andnecrosis-inducing effects of an extract according to the invention oncells corresponding to four different human cell lines, HCT-15 (FIG.17), A172 (FIG. 18), J82 (FIG. 19) and Hs683 (FIG. 20). The upper partof the figures shows apoptotic-inducing and necrosis-inducing effects ofthe said extract at different doses after 24 hours, the lower part ofthe figures after 72 hours.

FIG. 21 represents the effects of an extract according to the inventionon an in vivo lymphoma cancer model. The said extract was administeredby intra-peritoneal injection at doses of 2.5 mg/kg; 5 mg/kg and 10mg/kg extract in P388 lymphoma-tumor bearing mice. FIG. 21A representsthe effects of the said extract on the weight of control (untreated)mice and in treated mice. FIG. 21B represents the effects of the saidextract on the tumor growth in control mice and in treated mice.

FIG. 22 represents the effects of an extract according to the inventionon an in vivo lymphoma cancer model. The said extract was administeredby intra-peritoneal injection at doses of 0.63 mg/kg; 1.25 mg/kg and 2.5mg/kg extract in P388 lymphoma-tumor bearing mice. FIG. 22A representsthe effects of the said extract on the weight of control mice andtreated mice. FIG. 22B represents the effects of the said extract on thetumor growth in control mice and in treated mice

FIG. 23 represents the effects of an extract according to the inventionon an in vivo melanoma cancer model. The said extract was administeredby intra-peritoneal at doses of 2.5 mg/kg; 1.25 mg/kg and 0.63 mg/kgextract in B16-melanoma bearing mice. FIG. 23A represents the effects ofthe said extract on the weight of control (untreated) mice and treatedmice. FIG. 23B represents the effects of the said extract on the tumorgrowth in control mice and in treated mice.

FIG. 24 represents the effects of an extract according to the inventionon an in vivo melanoma cancer model. The extract was administered per osat doses of 0.63 mg/kg; 1.25 mg/kg and 2.5 mg/kg extract in B16-melanomabearing mice. FIG. 24A represents the effects of the said extract on theweight of control mice and treated mice. FIG. 24B represents the effectsof the said extract on the tumor growth in control mice and in treatedmice.

FIG. 25 represents the effects of an extract according to the inventionon an in vivo breast cancer model. The said extract was administered byintra-peritoneal injection at doses of 2.5 mg/kg; 5 mg/kg and 10 mg/kgextract in MXT-HI breast cancer bearing mice. FIG. 25A represents theeffects of the said extract on the weight of control (untreated) miceand treated mice. FIG. 25B represents the effects of the said extract onthe tumor growth in control mice and treated mice.

FIG. 26 represents the statistical general fit for the MXT-HI breastcancer bearing mice of the test groups (i.e. intra-peritoneal injectionof the mice at extract doses of 2.5 mg/kg; 5 mg/kg and 10 mg/kg) and thecontrol group, with regard to survival (Kaplan-Meier statisticalanalysis).

FIG. 27 represents the effects of an extract according to the inventionat doses of 5 mg/kg and 1.25 mg/kg on the amount of white blood cell,red blood cell, the hemoglobin and hematocrite concentration at daythree after intra-peritoneal injection in mice compared to control mice(In the Figure the ‘white’ extract dose is 1,25 mg/kg

FIGS. 28 to 35 illustrate the anti-poisonous effects of extractsaccording to the invention when combined with anti-tumors compoundsadriamycine or vincristine or camptothecin or oxaliplatin.

Properties of the Extract

The Calotropis procera plant, belonging to the family of theAsclepiadaceae, is a plant growing in Africa and Asia. This plant isused in traditional folk medicine and has also been studied with respectto a considerable number of uses such as an anti-pyretic, anti-malarial,anti-diarroeal, analgesic, anti-inflammtory gastric, mucosal protectorand as an insecticidal, anti-tussive, antibacterial, wound healing,muscle relaxant. The stems, flowers and leaves of Calotropis proceraplant are known to contain certain compounds known as cardenolides.Recently, cardiotoxic activity has been attributed to thesecardenolides, and they are exploited in human therapies for treatingcardiac insufficiencies.

Unexpectedly, the present invention relates to at least one extractaccording to the invention of the Calotropis procera plant showinganother type of activity, in particular an “anti-tumor activity”.Moreover, it was demonstrated that at least one extract according to theinvention of shows in vitro as well as in vivo anti-tumor effects. Thesaid extract according to the invention induces a dramatic reduction incell growth and shows a pro-apoptotic activity. These properties arefurther illustrated in the examples 3 and 4. In addition, it has beenin-vivo shown by the inventors that at least one of the said extractsaccording to the invention is highly active against several types ofcancers. As is shown in the examples described below, the said extractaccording to the invention, exerts significant anti-tumor effects onseveral tumor models tested, which represent a broad panel ofhistological tumor types, including breast cancer, lymphomas, melanomas.These models are clinically relevant because they mimic specificclinical stages of human cancers.

Surprisingly, the said extract according to the invention is highlyactive at relatively low doses. The said extract can exert an anti-tumoractivity at low doses in the range of 0.4 to 2.2 mg/kg and preferably inthe range of 0.5 to 2 mg/kg. The high anti-tumor activity at low dosesindicates that the said extract is highly active. Surprisingly, as willalso become clear when reading the examples given below, the saidextract according to the invention shows significantly higher anti-tumoreffects when assayed at chronically low doses, i.e. 0.6 mg/kg to 1.25mg/kg, than at high doses, i.e. at doses of 5 mg/kg to 10 mg/kg. TheMaximum Tolerated Dose (MTD) index of the said extract according to theinvention, referring to the maximum amount of the said extract, whichcan be administered acutely to healthy animals, is 20 mg/kg extract.This value indicates that the said extract according to the inventionhas a broad therapeutic window.

The terms “toxicity” or “toxic effects” relate to the detrimentaleffect(s) a compound may have on healthy cells, tissues or organs.Another important property of an extract according to the inventionincludes its low toxicity level. It was demonstrated that the saidextract does not induce in vivo hematological changes, does not induceweight loss and shows minor side effects on different types of organsand tissues. In fact, the toxicity level of the said extract accordingto the invention is surprisingly low. The said extract according to theinvention combines the essential features of a good anti-tumor activity,a low level of toxicity and a minimal induction of detrimental sideeffects.

Furthermore, the extracts of the plant Calotropis procera according tothe invention also show an “anti-poisonous activity”. As mentionedabove, the term “anti-poisonous activity”, as used herein, refers to theability of the extracts according to the invention to attenuate orreverse the detrimental side effects of compounds. Surprisingly, theinventors have demonstrated that combination of an extract according tothe invention with other therapeutic compounds, having detrimental sideeffects enables undesirable side effects of the therapeutic compound tobe reduced. For instance, example 8 illustrates that the combination ofan extract according to the invention with well known anti-cancercompounds, such as vincristine or adriamycine, camptothecin oroxaliplatin, enables the reduction of some side effects caused by thetoxicity to normal cells induced by these anti-cancer compounds.

The anti-poisonous activity is evident when at least one of the extractsaccording to the invention is administered prior to the therapeuticcompound i.e. they are administered sequentially. It is an aspect of theinvention to administer an extract prior any time before the therapeuticcompound is administered. It is a further aspect of the invention toadminister an extract no more than 336, 312, 288, 264, 240, 216, 192,168, 144, 120, 96, 72, 48, 24, 20, 16, 12, 8, 4, 2, 1, or 0.5 hoursbefore the therapeutic compound is administered.

The anti-poisonous activity is also evident when at least one of theextracts according to the invention is administered at the same time asthe therapeutic compound i.e. as a composition, by simultaneous orseparate administration.

The anti-poisonous activity is also evident when at least one of theextracts according to the invention is administered after thetherapeutic compound i.e. they are administered sequentially. It is anaspect of the invention to administer an extract any time after thetherapeutic compound has been administered. It is a further aspect ofthe invention to administer the extract less than 0.5, 1, 2, 4, 8, 12,16, 20, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, or366 hours after the therapeutic compound has been administered.

In sequential administration, the said extracts may be administeredonce, or any number of times and in various doses before and/or afteradministration of the therapeutic compound. Sequential administrationmay be combined with simultaneous or sequential administration.

Composition of the Extract

The extracts according to the invention comprise one or several activecompounds, i.e. compounds present in an extract according to theinvention, which exhibit an activity similar to at least one of theabove-defined activities of an extract according to the invention. Themain activities of at least one of the extracts are an anti-tumoractivity and an anti-poisonous activity. As it will be understood by theone skilled in the art, an active compound present in the said extractsmay exerts only one of these properties, or even both of theseproperties.

In another embodiment, at least one of the extracts according to theinvention comprise at least two of the active compounds selected fromthe group comprising asclepin, calactin, voruscharin, calotropin,calotropagenin, uzarigenin, calotoxin, uscharin and uscharidin, 2″oxovoruscharin.

As it will be understood, the extracts according to the invention mayfurther contain one or more active compounds, which do not belong to thegroup of the cardenolides, and other compounds. In another embodiment,the extracts contain at least one of the compounds represented inTable 1. TABLE 1 Some of the compounds contained in at least one of theextracts according to the invention Compounds present3-O-ACETYL-CALOTROPIN, ALPHA-AMYRIN, in the extractALPHA-AMYRIN-BENZOATE, ALPHA- according to the CALOTROPEOL,ALPHA-LACTUCEROL, invention ALPHA-LACTUCERYL-ACETATE, ALPHA-LACTUCERYL-ISOVALERATE, ARABINOSE, ASH, BENZOYLISOLINEOLONE, BENZOYLLI-NEOLONE, BETA-AMYRIN, BETA-AMYRIN- BENZOATE, BETA-CALOTROPEOL, BETA-SITOSTEROL, CAOUTCHOUC, COROGLAUCO- GENIN; COROTOXIGENIN; D-GLUCOSAMINE,FRUGOSIDE, GIGANTEOL, GIGANTIN, GLUCOSE, HISTAMINE, ISOGIGANTEOL,ISOLACTUCEROL, ISOLINEOLONE, LAURANE LINEOLONE, LINOLEIC-ACID,LINOLENIC- ACID, MELISSYL-ALCOHOL, MUDARINE, OLEIC- ACID, PALMITIC-ACID,PROCERO- SIDE, PSEUDOCALOTROPAGENIN, RHAMNOSE STIGMASTEROL, SYRIOGENINTARAXASTEROL, TARAXASTEROL- BENZOATE, TRYPSIN

In another embodiment, the present invention relates to an activecompound isolated from the extracts according to the invention.

In another embodiment of the invention, an active compound of theextract is 2″oxo-voruscharin. An aspect of the invention is theanti-cancer and/or anti-poisonous activity of said compound (compound A)and the derivatives (compounds B to H) thereof as shown in Table 2.TABLE 2 Chemical structure of 2″oxo-voruscharin (Compound A) andderivatives there of (Compounds B to H). Formula I

R₁ R₂ R₃ R₄ R₅ Compound A —COH —OH —OH ═O —H 2″oxo-voruscharin CompoundB —CH₂OH —OH —OH ═O —H Compound C —CH₂OAc —OH —OH ═O —H Compound D—CH₂OOCphenyl —OH —OH ═O —H Compound E —CH₂OH —OH —OH Double bond* —HCompound F —CH₂OAc —OH —OH Double bond* —H Compound G —CH₂OOCphenyl —OH—OH Double bond* —H Compound H —CH₂OH —OH —OH

—H

-   -   means a double bond between the N atom and the C carbon atom of        the N-containing heterocyclic ring of formula 1.        Methods of Extraction

According to another embodiment, an extract according to the inventioncan be obtained by an alcoholic extraction, in particular by a methanolextraction.

In another embodiment, the present invention relates to a method forobtaining an extract according to the invention comprising the steps of:

-   -   a) extracting the starting material of said Calotropis procera        plant, said starting material being selected among fruits,        aerial parts, subterranean parts, and their mixtures, in an        aliphatic alcohol, by dissolving the starting material in said        alcohol thereby obtaining a suspension of said material in said        alcohol, stirring said suspension; and filtering said suspension        by fritted glass thereby obtaining a first filtrate and a first        solid part;    -   b) extracting said first solid part in an aliphatic alcohol        thereby obtaining a second filtrate and a second solid part;    -   c) combining said first and said second filtrate thereby        obtaining a combined filtrate; and    -   d) evaporating said combined filtrate under vacuum thereby        obtaining an oily residue.

In a preferred embodiment, the starting material of said Calotropisprocera plant is selected from subterranean parts, in particular fromroots.

In another embodiment, the aliphatic alcohol used to extract thestarting material according to the invention is methanol. In yet anotherembodiment, the residue obtained in the extraction process is taken upin a solvent, in particular in a pharmaceutically acceptable solvent.

In another embodiment, the present invention relates to a method forobtaining extracts according to the invention, comprising the steps of:

-   a) Grinding the starting material of leaf blades, stems, barks and    roots of Calotropis procera give a fine powder of the plant.-   b) Extracting the powder of step a) with dichloromethane for at    least 6, 12, 18 or preferably 24 hours using a soxhlet extractor-   c) decanting the dichloromethane of step b), and evaporating the    filtrate, after filtration, to form a gum-   d) extracting the residue of step c) with methanol for at least 6,    12, 18 or preferably 24 hours using a soxhlet extractor-   e) decanting the methanol of step d), evaporating the filtrate,    after filtration, to form a gum-   f) subjecting the gum of step e) to column chromatography using    flash silica gel and dichloromethane-methanol as solvent-   g) collecting a first fraction,    -   h) applying the concentrated fraction of step g) to column        chromatography using flash silica gel and hexane-acetone as        solvent to give two fractions,-   i) washing the column after step h) with methanol to give a third    fraction,

Extracts according to the present invention include those obtained fromstep c), step g), and step i).

It is an aspect of the invention that the extractions are performed in amanner compatible with preserving the integrity of the biologicallyactive compounds contained in said extract. Such precautions are knownto the skilled person.

Applicability of the Extracts

Due to the interesting properties of at least one of the extractsdisclosed herein, in particular its anti-tumor activity, itsant-poisonous activity, its low level of toxicity and/or its highactivity at low doses, the extract(s) according to the invention oractive compounds thereof are particularly suitable for use as amedicament for the treatment of diseases, and in particular for treatingcancer.

In another embodiment, the invention relates to the use of an extractaccording to the invention, or an active compound thereof, as amedicament.

In yet another embodiment, the invention also relates to the use of atleast one extract according to the invention, or an active compoundthereof, for the preparation of a medicament in the treatment of cancer.In particular the said extract according to the invention, or an activecompound thereof is used for the preparation of a medicament in thetreatment of a cancers. Examples of cancers that may be treatedaccording to the invention include, but are not limited to breastcancer, lymphoma, sarcoma, pancreatic cancer, melanoma, colorectalcancer, glioma, non small cell lung cancer, small cell lung cancer, skincancer, bone cancer, ovarian cancer, CNS cancer, renal cancer, bladdercancer, head and neck cancer, prostate cancer, liver cancer andhematological cancer.

As mentioned above, the extracts according to the invention alsocomprise an anti-poisonous activity. The inventors have demonstratedthat a combination of an extract according to the invention or activecompounds thereof with a second compound having relevant detrimentalside effects enables a reduction of the toxic activity of this secondcompound, without reducing its therapeutic activity. Therefore, theCalotropis procera extracts according to the invention may also becombined in a medicament with other compounds, in particular with otheranti-cancer compounds.

Pharmaceutical Compositions Comprising the Extract

In another embodiment, the present invention relates to a pharmaceuticalcomposition for the treatment of cancer comprising a therapeuticeffective amount of an extract of Calotropis procera according to theinvention or an active compound thereof, and a pharmaceutical acceptablecarrier.

In another embodiment, the present invention relates to a pharmaceuticalcomposition for the treatment of cancer comprising

-   -   a therapeutic effective amount of an extract of Calotropis        procera according to the invention or an active compound        thereof,    -   a therapeutic effective amount of a therapeutic compound, and,    -   a pharmaceutical acceptable carrier.

The term “therapeutically effective amount” as used herein means thatamount of at least one extract or active compound or pharmaceuticalagent that elicits the biological or medicinal response in a tissue,system, animal or human that is being sought by a researcher,veterinarian, medical doctor or other clinician, which includesalleviation of the symptoms of the disease being treated.

The pharmaceutical composition can be prepared in a manner known to oneof skill in the art. For this purpose, an extract according to theinvention and/or any active compound thereof, one or more solid orliquid pharmaceutical carriers and, if desired, in combination withother pharmaceutical active compounds, are brought into a suitableadministration form or dosage form which can then be used as apharmaceutical in human medicine or veterinary medicine.

Particular forms of the pharmaceutical composition may be, for example,solutions, suspensions, emulsions, creams, tablets, capsules, nasalsprays, lipsomes or micro-reservoirs, especially compositions in orallyingestible or sterile injectable form, for example, as sterileinjectable aqueous or oleaginous suspensions or suppositories. Thepreferred form of composition contemplated is the dry solid form, whichincludes capsules, granules, tablets, pills, boluses and powders. Thesolid carrier may comprise one or more carriers, e.g. lactose, fillers,disintegrating agents, binders, e.g. cellulose, carboxymethylcelluloseor starch or anti-stick agents, e.g. magnesium stearate, to preventtablets from adhering to tabletting equipment Tablets, pills and bolusesmay be formed so as to disintegrate rapidly or to provide slow releaseof the active ingredient.

Furthermore, due to their anti-poisonous activity, the extractsaccording to the invention or active compounds thereof, are alsoparticularly suitable to be combined with other therapeutic compoundswhich exert a pharmacological activity having toxic side effects, suchas other medicaments, which show toxic side effects.

The “therapeutic compound, which exerts a pharmacological activityhaving toxic side effects” may include any compound that is used for thetreatment of any disease but which induces unwanted toxic effects.Preferably, such compounds are compounds that are used in the treatmentof cancer. For instance, an extract according to the invention or activecompounds thereof can be combined with one or more than one othercompound that has an anti-tumor effect. Since two or more compoundshaving an anti-tumor effect are combined, an improved anti-tumoractivity can be obtained. In addition, such combinations also enable toreduce the toxic side effects, which are induced by one or several ofthe anti-tumor compounds.

A combination of an extract according to the invention or activecompounds thereof with another therapeutic compound may involve aseparate use of the said extract or active compound thereof and theother therapeutic compound. In particular, the combination may involvethe use of an extract according to the invention or active compoundsthereof prior (pre-treatment) to, at the same time, or after(post-treatment) the use of the other therapeutic compound.

Alternatively, a combination of an extract or active compounds thereofwith another therapeutic compound may also involve a mixture of bothelements. Therefore, in a preferred embodiment, the present inventionrelates to a pharmaceutical composition comprising a first component,said first component being the above-described extract or at least oneactive compound thereof, and a second component, said second componentcomprising a therapeutic compound, which exerts a pharmacologicalactivity having toxic side effects.

Product

One aspect of the invention is a product containing an extract ofCalotropis procera and a therapeutic compound for simultaneous, separateor sequential administration to a subject. It is an aspect of theinvention that the product may be used according to the invention.

By simultaneous administration means the two components are administeredto a subject at the same time. For example, as a mixture of the twocomponents. Examples include, but are not limited to a solutionadministered intraveneously, a tablet, liquid, topical cream, etc.,wherein each preparation comprises both components.

By separate administration means the two components are administered toa subject at the same time or substantially the same time, but thecomponents are present in the product as separate, unmixed preparations.For example, the two components may be present in the product asindividual tablets. The tablets may be administered to the subject byswallowing both tablets at the same time, or one tablet directlyfollowing the other.

By sequential administration means the two components are administeredto a subject sequentially and the components are present in the productas separate, unmixed preparations. There is a time interval betweendoses. For example, one component might be administered up to 336, 312,288, 264, 240, 216, 192, 168, 144, 120, 96, 72, 48, 24, 20, 16, 12, 8,4, 2, 1, or 0.5 hours after the other component.

In sequential administration, the extract may be administered once, orany number of times and in various doses before and/or afteradministration of the therapeutic compound. Sequential administrationmay be combined with simultaneous or sequential administration.

Kit

Another aspect of the present invention is a kit comprising a containerin which an extract of Calotropis procera is present, and a container inwhich a therapeutic compound is present. An extract according to theinvention and therapeutic compound according to the invention aredescribed above.

By mentioning a kit comprising a container in which an extract ofCalotropis procera is present, and a container in which a therapeuticcompound, it is to be understood that the kit may contain a single dose,or a plurality of doses, reflected in two single containers, two singlecontainers each capable of holding a plurality of doses or a pluralityof single containers each capable of holding a single dose. It is to beunderstood that the kit may comprise more than one therapeutic compound,each therapeutic compound in a separate container, or a mixture oftherapeutic compounds in a single container.

The containers may be any known in the art of medicines. They may bevials, blister packs, syringes, resealable bottles, bottles withdispensing means, aspirators, dropping bottles, patches, applicators,suppositories.

The separate containers enable to extract and therapeutic compound to beadministered simultaneously, separately, or sequentially.

Method of Treatment

Due to the favorable anti-tumor properties of at least one of theextracts according to the present invention, said extracts areparticularly useful in the treatment of individuals suffering fromcancer. In another embodiment, the present invention also relates to amethod of treatment of cancer comprising administering to an individualin need of such treatment a pharmaceutical composition according to theinvention. Due to its low level of toxicity and its minimal sideeffects, use of one said extract in a pharmaceutical composition for thetreatment of cancer will involve minimal side effects. As a consequence,a said the extract according to the invention may be used during alonger period of time during the treatment of cancer.

In particular, in a preferred embodiment, the invention relates to amethod for treating cancer, wherein the cancer is selected from thegroup comprising, but not limited to breast cancer, lymphoma, sarcoma,pancreatic cancer, melanoma, colorectal cancer, glioma, non small celllung cancer, small cell lung cancer, skin cancer, bone cancer, ovariancancer, CNS cancer, renal cancer, bladder cancer, head and neck cancer,prostate cancer, liver cancer and hematological cancer.

For these purposes, the pharmaceutical composition of the presentinvention may be administered orally, parenterally, i.e. includingsubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques, by inhalation spray, or rectally, indosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles.

In accordance with the method of the present invention, saidpharmaceutical composition can be administered separately at differenttimes during the course of therapy or concurrently in divided or singlecombination forms. The present invention is therefore to be understoodas embracing all such regimes of simultaneous or alternating treatmentand the term “administering” is to be interpreted accordingly.

Essentially, the primary modes of treatment of solid tumor cancerscomprise surgery, radiation therapy and chemotherapy, separately and incombination. The extracts according to the invention are suitable foruse in combination with these medicinal techniques. The extractsaccording to the invention may be useful in increasing the sensitivityof tumor cells to radiation in radiotherapy and also in potentiating orenhancing damage to tumors by chemotherapeutic agents. The extractsaccording to the invention may also be useful for sensitizingmultidrug-resistant tumor cells. The extracts according to the inventionare useful therapeutic compounds for administration in conjunction withother DNA-damaging therapeutic compounds including radiation used inradiotherapy to potentiate their effect.

It will be understood, that the pharmaceutical composition according tothe invention can be administered to humans in specific dose levels andat specific frequency of dosage which may be varied for any particularpatient and which will depend upon a variety of factors including theage, body weight, general health, sex, diet, mode and time ofadministration, rate of excretion, drug combination, the severity of theparticular condition of the patient undergoing therapy.

The following examples are meant to illustrate the present invention.These examples are not to be considered as limiting the scope of theinvention. A first example illustrates the extraction of extractsaccording to the invention form the plant Calotropis procera. Examples 2to 4 relate to the in vitro anti-tumor characterization of an extractaccording to the invention. Examples 5 to 7 describe the in vivoanti-tumor characterization of an extract according to the invention.Example 8 illustrates the anti-poisonous effects of extracts accordingto the invention.

EXAMPLES Example 1 Extraction Process of Extracts According to theInvention from the Plant Calotropis procera

An extract (Extract A) according to the invention was isolated form theroots of Calotropis procera plant (Asclepiadiaceae family) by methanol.The said extract is further referred as Extract A (to adapt). About 10grams of plant was put in an erlenmeyer with 150 ml of methanol. Thesuspension was shaken magnetically for 12 hours and then filtered on aglass frit. The remaining solid was extracted a second time by methanolfor 2 hours. Both filtrates were combined and evaporated under vacuum byusing rotavapor. The residue constituted the methanolic extract isreferred hereafter as Extract A.

Analysis of Extract A revealed the presence of several types ofcompounds. A particular group of compounds identified in said Extract Acomprises cardenolides such as asclepin, calactin, vorusharin,calotropin, calotropagenin, uzarigenin, usharin and usharidin. Inaddition, some other compounds, present in Extract A comprise, but arenot limited to, 3-O-ACETYL-CALOTROPIN, ALPHA-AMYRIN,ALPHA-AMYRIN-BENZOATE, ALPHA-CALOTROPEOL, ALPHA-LACTUCEROL,ALPHA-LACTUCERYL-ACETATE, ALPHA-LACTUCERYL-ISOVALERATE, ARABINOSE,BENZOYLISOLINEOLONE, BENZOYLLINEOLONE, BETA-AMYRIN,BETA-AMYRIN-BENZOATE, BETA-CALOTROPEOL, BETA-SITOSTEROL, CAOUTCHOUC,COROGLAUCOGENIN; COROTOXIGENIN; D-GLUCOSAMINE, FRUGOSIDE, GIGANTEOL,GIGANTIN, GLUCOSE, HISTAMINE, ISOGIGANTEOL, ISOLACTUCEROL, ISOLINEOLONE,LAURANE LINEOLONE, LINOLEIGACID, LINOLENIC-ACID, MELISSYL-ALCOHOL,MUDARINE, OLEIC-ACID, PALMITIC-ACID, PROCEROSIDE, PSEUDOCALOTROPAGENIN,RHAMNOSE STIGMASTEROL, SYRIOGENIN TARAXASTEROL, TARAXASTEROL-BENZOATEand TRYPSIN.

Other extracts according to the invention, where isolated by a method,comprising the steps of:

-   a) Grinding the starting material of leaf blades, stems, barks and    roots of Calotropis procera to give a fine powder of the plant,-   b) Extracting the powder of step a) with dichloromethane for 24    hours using a soxhlet extractor,-   c) decanting the dichloromethane of step b), and evaporating the    filtrate (after filtration) to form a gum,-   d) extracting the residue of step c) with methanol for 24 hours    using a soxhlet extractor,-   e) decanting the methanol of step d), evaporating the filtrate    (after filtration) to form a gum,-   f) subjecting the gum of step e) to column chromatography using    flash silica gel and dichloromethane-methanol as solvent,-   g) collecting a first fraction,-   h) applying the concentrated fraction of step g) to column    chromatography using flash silica gel and hexane-acetone as solvent    to give two fractions,-   i) washing the column after step h) with methanol to give a third    fraction.

The extract isolated at step c) is referred hereafter as Extract B. Theextract isolated at step g) is referred hereafter as Extract C. Theextract isolated at step i) is referred hereafter as extract D.

Example 2 Effect of an Extract According to the Invention on OverallCell Growth of a Cell Line

This example illustrates the anti-tumor activities of Extract Aaccording to the invention on different types of cancer.

In order to characterize the in vitro activities of the Extract A, MTTtests were carried out. The MTT test, which is a well known test in theart, is an indirect technique that rapidly measures, i.e. within 5 days,the effect of a given product on the overall growth of a cell line. Thistest measures the number of metabolically active living cells that areable to transform the MTT product (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide), having a yellowish color, to the blueproduct formazan by mitochondrial reduction only performed by livingcells. The amount of formazan obtained at the end of the experiment ismeasured with a spectrophotometer and is directly proportional to thenumber of living cells.

Forty-eight human cancer cell lines, described in Table 2, were testedin the presence of Extract A. These cell lines covered ten histologicaltypes, being pancreatic cancer, sarcoma, breast cancer, melanoma, coloncancer, glioma, lung cancer, bladder cancer, prostate cancer and headand neck cancer. The cells were allowed to grow in flat bottomed 96-wellmicro-wells with 100 μl of cell suspension per well and between 3,000and 5,000 cells/well depending on cell type. Each cell line was seededin its own cell culture medium as indicated in Table 2. TABLE 2 Humancancer cell lines and corresponding cell culture medium used for the MTTexperiments Cell lines ATCC code Tissue Medium BxPC-3 CRL-1687Pancreatic RPMI 10% serum MiaPACA-2 CRL-1420 Pancreatic DMEM glucose 10%PANC-1 CRL-1469 Pancreatic OPTIMEM 5% CAPAN-1 HTB-79 Pancreatic RPMI 10%serum CFPAC-1 CRL-1918 Pancreatic Iscove's 10% serum Hs766T HTB-134Pancreatic OPTIMEM 5% SU.86.86 CRL-1837 Pancreatic RPMI 10% serumSK-LMS-1 HTB-88 Sarcoma MEM 10% serum SK-UT-1B HTB-115 Sarcoma MEM 10%serum AA HT-1080 CCL-121 Sarcoma MEM 5% serum Hs729 HTB-153 Sarcoma MEM10% serum AA MES-SA CRL-1976 Sarcoma MEM 5% serum RD CCL-136 Sarcoma MEM5% serum A204 HTB-82 Sarcoma MEM 5% serum MCF-7 HTB-22 Breast MEM 5%serum T-47D HTB-133 Breast MEM 5% serum MDA-MB-231 HTB-26 Breast DMEMNut mix 10% serum ZR-75-1 CRL-1500 Breast MEM 5% serum Hs578T HTB-126Breast MEM 5% serum SK-MEL-28 HTB-72 Melanoma RPMI 10% serum HT-144HTB-63 Melanoma MEM 10% serum AA C-32 CRL-1585 Melanoma MEM 5% serumMalme-3M HTB-64 Melanoma MEM 5% serum G-361 CRL-1424 Melanoma OPTIMEM 5%serum HCT-15 CCL-225 Colon MEM 5% serum LoVo CCL-229 Colon MEM 5% serumDLD-1 CCL-221 Colon MEM 10% serum AA Ls-174T CL-188 Colon MEM 10% serumAA HT29 HTB-38 Colon MEM 10% serum AA WIDR CCL-218 Colon MEM 10% serumAA SW948 CCL-237 Colon OPTMEM 5% serum A172 CRL-1620 Glioma MEM 5% serumH4 HTB-148 Glioma MEM 5% serum Hs683 HTB-138 Glioma MEM 5% serum SW1088HTB-12 Glioma MEM 5% serum U-118MG HTB-15 Glioma MEM 5% serum U-87MGHTB-14 Glioma MEM 5% serum U-373MG HTB-17 Glioma MEM 5% serum A427HTB-53 Lung MEM 5% serum A549 CCL-185 Lung MEM 5% serum J82 HTB-1Bladder MEM 5% serum T24 HTB-4 Bladder MEM 5% serum PC3 CRL-1435Prostate MEM 5% serum Detroit CCL-138 Head and neck MEM 10% serum AARPMI CCL-30 Head and neck MEM 10% serum AA FaDu HTB-43 Head and neck MEM10% serum AA SCC9 CRL-1629 Head and neck MEM 10% serum AA SCC25 CRL-1628Head and neck MEM 10% serum AAWhere AA means “Amino Acids”

After a 24-hour incubation period at 37° C. the culture medium wasreplaced by 100 μl of fresh medium in which Extract A was dissolved atthe different concentrations of 0.01 μg/ml, 0.05 μg/ml, 0.1 μg/ml, 0.5μg/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml, 50 μg/ml and 100 μg/ml. Eachexperimental condition was carried out in sextuplicate.

After 72 hours of incubation at 37° C. with the drug, i.e. experimentalconditions or without the drug, i.e. control, the medium was replaced by100 μl MTT at the concentration of 1 mg/ml dissolved in RPMI. Themicro-wells were then incubated for 3 hours at 37° C. and centrifuged at400 g for 10 minutes. The MTT was removed and the formed formazancrystals were dissolved in 100 μl DMSO. The micro-wells were shaken for5 minutes and read on a spectrophotometer at 2 wavelengths at 570 nmcorresponding to the maximum formazan absorbance wavelength, and at 630nm, which is the background noise wavelength.

For each experimental condition, the mean OD associated with thestandard error of the mean (SEM) for each condition, i.e. 6 wells, wascalculated. The percentage of remaining living cells was calculated incomparison with control. Results of these experiments are represented inFIGS. 1 to 11.

FIG. 1 represents the overall results for the ten histological types. Asindicated Extract A exerted an anti-tumor effect on all histologicaltypes tested. The human tumor cell lines issued from bladder presented asensitivity to Extract A which was weaker than the remaining ninehistological types. The concentration at which the plant extract killed50% of the cells population, the so-called IC₅₀ value, was determined.Said IC₅₀ value comprised between 0.5 μg/ml and 1 μg/ml for the celllines issued from the breast and the pancreas cell lines. A moreimportant anti-tumor effect with a IC₅₀ value of 0.5-0.1 μg/ml wasobtained for sarcoma, melanoma, glioma, lung, head and neck andcolorectal, cancer cell lines. The PC3 prostate cancer cells exhibited amarked sensitivity, with an IC₅₀ value in range of 0.01 to 0.05 μg/ml.

As illustrated on FIG. 1, the mean IC₅₀ value of the 5 breast cancercell lines ranged between 0.5 and 1 μg/ml. The overall growth of the 5individual breast cancer cell lines, T-47D, MDA-MB-231, ZR-75-1, MCF-7and Hs578T, is further illustrated in FIG. 2. The cell lines T-47D,MDA-MB-231, ZR-75-1 and Hs578T exhibited similar sensitivities toExtract A, while MCF-7 cells had their overall growth reduced morerapidly than the remaining four breast cancer models.

As illustrated on FIG. 1, the mean IC₅₀ value of the 7 sarcoma cancercell lines (see Table 2) ranged between 0.5 and 0.1 μg/ml. The overallgrowth of the individual cell lines is further illustrated in FIG. 3.Extract A induced the most important anti-tumor effect on the A204 cellline. In fact, the IC₅₀ value appeared to be between 0.1 and 0.05 μg/ml.The Hs729 cell line was considered to the least sensitive of the 7sarcoma cell lines. It nevertheless showed IC₅₀ values for Extract Aranging between 1 and 5 μg/ml.

As illustrated in FIG. 1, the mean IC₅₀ value of the 7 pancreatic cancercell lines (see Table 2) ranged between 0.5 and 0.1 μg/ml. Similaroverall growths were observed for 6 of the 7 pancreatic cancer celllines (FIG. 4). Of these 7 cell lines, Panc-1 seemed to be the mostsensitive to Extract A and Hs766T the least. The IC₅₀ values of these 2lines ranged between 0.1 and 0.5 μg/ml, and between 1 and 5 μg/mlrespectively.

As illustrated on FIG. 1, the mean IC₅₀ value of the 5 melanoma cancercell lines (see Table 2) ranged between 0.5 and 0.1 μg/ml. The extractaccording to the invention reduced the overall growth of all themelanoma cell lines by more than 60% at concentrations equal to orhigher than 5 μg/ml (FIG. 5). Malme-3M and G-361 were the most sensitiveof the 5 melanoma cell lines. They exhibited a similar overall growth,with the IC₅₀ value ranging between 0.1 and 0.5 μg/ml.

As illustrated on FIG. 1, the mean IC₅₀ value of the 7 colon cancer celllines (see Table 2) ranged between 0.5 and 0.1 μg/ml. Of the differentcolorectal cancer cell lines tested (FIG. 6) the LoVo line had an IC₅₀value of around 0.1 μg/ml and was considered to be the most sensitivecolorectal line. The SW948 tumor cell line was considered to be theleast sensitive, with a IC₅₀ value ranging between 0.5 and 1 μg/ml.

As illustrated in FIG. 1, the mean IC₅₀ value of the 7 human gliomacancer cell lines (see Table 2) ranged between 0.5 and 0.1 μg/ml. Hs683was the most sensitive cell line to extract (FIG. 7). Its IC₅₀ value wasnear 0.05 μg/ml. In contrast, the growth of A172 cells was not affectedby Extract A at concentrations from 0.01 to 10 μg/ml. The IC₅₀ valueranged between 10 and 50 μg/ml.

As illustrated on FIG. 1, the mean IC₅₀ value of the 2 humannon-small-cell-lung cancer cell lines (NSCLC) ranged between 0.1 and 0.5μg/ml. FIG. 8 shows that both lines were sensitive to Extract A.

As illustrated on FIG. 1, the mean IC₅₀ value of the 2 human bladdercancer cell lines (see Table 2) ranged between 50 and 100 μg/ml. The 2lines exhibited marked differences in term of sensitivity to Extract A(FIG. 9). In fact, at 5 μg/ml of Extract A reduced the overall growth ofthe T24 cell line by more than 80% while the overall growth of the J82cell line was only decreased by 32% at 100 μg/ml. The J82 cell lineseemed very weakly sensitive to Extract A.

The overall growth of 1 prostate cancer cell line (the PC3 cell line)was evaluated by MTT assay when the cells had been treated with ExtractA. Extract A induced an inhibition of overall growth of about 90% atbetween 100 μg/ml and 0.5 μg/ml. The IC₅₀ value was around 0.1 μg/ml(FIG. 10).

As illustrated on FIG. 1, the mean IC₅₀ value of the 5 head and neckcancer cell lines ranged between 0.1 and 0.5 μg/ml. Similar overallgrowths were observed for 4 of the 5 head and neck cancer cell lines(FIG. 11). Of these 5 cell lines, SCC9 seemed to be the least sensitive.It nevertheless showed IC₅₀ values for Extract A of around 5 μg/ml.

Summarized, Extracts A according to the invention exerts a dramaticanti-tumor effect on forty-six of the forty-eight human cancer celllines assayed in the experiments described above. These anti-tumoreffects correspond to marked decreases in the overall growth of thesehuman cancer models representing a very broad panel of histologicaltypes.

Example 3 Effect of an Extract According to the Invention on CellKinetics

This example illustrates the cytostatic effect of Extract A according tothe invention. According to the experiments performed by means of theMTT calorimetric assay described in example 2, it is clear that ExtractA dramatically decreases the overall growth of most of the fourty-eighthuman cancer cell lines submitted to the MTT assay. In the followingexamples it was investigated whether this extract induced decrease inoverall growth corresponds either to modifications occurring at thelevels of the cell cycle kinetics (example 3), or the induction ofapoptosis (see example 4), or of both.

The cell cycle is in general divided in several phases comprising aG0/G1, S and G2/M phase. Modifications taking place around the proteinscontrolling cell proliferation and/or cell death may constitute onetarget of the active compounds, present in Extract A. Modifications tocell cycle kinetics can be investigated by means of flow cytometry usingdifferent fluorophores f.e. propidium iodide, orange acridine andethidium bromide etc. Flow cytometry enables each cancer cell (runningto several thousands) to be located into the cell cycle.

Changes in the DNA histogram pattern are thus used to characterize themechanism of action of various therapeutic compounds. The data resultingfrom the flow cytometry analysis is processed graphically ormathematically in order to derive meaningful estimates of the G1, the Sand the G2/M compartments. Most mathematical processing is based oncertain models and assumptions.

Cell lines were seeded in flasks (25 cm² area) containing 7 ml ofculture medium. After 48 hours incubation at 37° C. the cell culturemedium was replaced by fresh medium in which Extract A had beendissolved at concentrations which kill 50%, (IC₅₀), 30% (=IC₃₀) and 10%(IC₁₀) of the cell population. After 24 or 72 hours of treatment thecells were harvested in suspension, washed in Phosphate Buffer Saline(PBS) at 4° C. and permeabilized with 70% ethanol (at 4° C.) overnightat −20° C. The cells were then washed with PBS and incubated withpropidium iodide solution (80 μg/ml) for 30 minutes at 37° C. andafterwards kept at 4° C. overnight. Ribonuclease A (3% V/V) was added toinduce a double-stranded DNA break. The portrait of the cell cycle wasestablished for each sample. A specific software program incorporatedinto the flow cytometer was used to define precisely the percentage ofcells in the different cell cycle phases. Each cell cycle phase wasreported in terms of peak surface and calculated as a percentage. Thesurface of the entire cell cycle was 100%. Each experiment was carriedout 3 times. The mean percentage of each different phase and thestandard error of the related mean was calculated. Each cell cycle phaseof a given condition was compared with the same cell cycle phase ofcontrol cells, i.e non-treated cells.

Extract A is highly effective against human tumor cell lines. Theconcentrations used in our flow cytometry experiments were chosen inaccordance with the MTT results (example 2). The five human cancer celllines Hs683 (glioma), J82 (bladder) A172 (glioma), RPMI (head-and-neck)and HCT-15 (colon) were tested and doses that killed 10, 30 and 50percent of the cells were determined (Table 3) in order to investigatewhether Extract A promoted an accumulation in one of the cell cyclephases when the cultures were treated for 24 or 72 hours with increasingconcentrations of Extract A. TABLE 3 Doses of Extract A according to theinvention killing 10, 30 and 50% of the cells in five cell lines Celllines IC₅₀ IC₃₀ IC₁₀ HCT-15 0.25 μg/ml 0.1 μg/ml 0.05 μg/ml RPMI 0.25μg/ml 0.1 μg/ml 0.05 μg/ml A172 25 μg/ml — 10 μg/ml J82 — 50 μg/ml 10μg/ml Hs683 0.05 μg/ml — 0.025 μg/ml

The HCT-15 cell cycle analyses (FIG. 12) showed that the distribution ofthe cell population was similar to the control after 24 hours oftreatment independent of the concentrations tested. On the other hand, aprominent S-population appeared in the cells treated with 0.25 μg/mlextract for 72 hours. The S fraction represented 21% of the cells incontrol and reached 59% upon treatment with Extract A at 0.25 μg/ml. Theincrease in the S fraction was accompanied by a loss of cells in theG0/G1 phase. The G0/G1 population underwent a marked decrease from 71%to 29%.

The analyses of the RPMI cell cycle (FIG. 13) showed that thedistribution of the cell population treated with Extract A at 0,05 μg/mlwas similar to the control after 24 hours of treatment. A weak increasein phase S was observed at 0,1 and 0,25 μg/ml. In contrast, an largeincrease in the S-population occurred when the RPMI cell line wasincubated for 72 hours with Extract A at the three concentrationstested. There were about 71%, 69% and 62% of cells in the S-phase whenExtract A was assayed at 0.05 μg/ml, 0.1 μg/ml and 0.25 μg/ml,respectively. Concomitantly, the percentage of cells in the G0/G1 phasedecreased markedly and the G2/M phase increased slightly, reaching 29%of the cell population.

The A172 cell line was treated with Extract A at 10 and 25 μg/ml, whichcorresponds to the IC₁₀ and IC₅₀. The results (FIG. 14) showed thatwhatever the concentrations tested Extract A induced an increase in theG0/G1 phase after 24 hours of treatment. Thirty-nine percent of thecells were in the G0/G1 phase in control while the cells treated withExtract A reached 60%. Concomitantly with the accumulation in the G0/G1phase, we observed a slight accumulation in the G2/M phase at thegreatest concentration tested. The effect observed after 24 hours oftreatment disappeared after 72 hours. Indeed, no significant change wasobserved in the different cell cycle phases.

The J82 cell line is hardly sensitive to the extract (cfr. MTT assay).The concentrations chosen for flow cytometry corresponded to about IC₁₀and IC₃₀ (FIG. 15). After 24 hours of treatment a slight accumulation inG0/G1 phase was obtained whatever the concentration tested. On the otherhand, 30 and 47% respectively of the cell population was in the S phase,when the cell were treated for 72 hours with Extract A at 50 and 100μg/ml while the control condition reached only 18%. At 100 μg/ml, anaccumulation in G2/M phase could also be observed.

After 24 hours of treatment with Extract A at 0.05 μg/ml the Hs683 cellshad accumulated in the G0/G1 phase and attained 70% as compared tocontrol (58%) (FIG. 16). We observed concomitantly a decrease in thepercentage of cells in the S phase. In the same way, at the bothconcentrations most of the cells were in the G0/G1 phase after 72 hoursof treatment. Indeed, more than 60% of the cells were in this phasewhile the cells in this phase in control represented 46% of cellpopulation.

In conclusion, Extract A principally induces an accumulation in theG0/G1 phase and under such circumstances this indicates that Extract Ahas a cytostatic effect.

Example 4 Pro-Apoptotic Effect of an Extract According to the Invention

This example illustrates the pro-apoptotic effect of Extract A accordingto the invention.

Cell death may occur in two different ways, either accidentally or asgenetically programmed. Accidental cell death, also referred to asnecrosis essentially occurs as the results of e.g. physical orbiological aggression. Apoptosis refers to the form of cell death whichis genetically programmed cell death and which occurs under normalphysiological conditions. After the induction of apoptosis, a cascade ofevents is induced in the cell, which comprises the activation of celldeath receptors, the activation of a serie of cytosolic proteases, theformation of apoptotic bodies, the fragmentation of the DNA.

The effect of Extract A on the apoptosis pathway was investigated on thefour human cancer cell lines: Hs683 (glioma), J82 (bladder), A172(glioma) and HCT-15 (colon).

Cells were seeded in flasks (25 cm² area) containing 7 ml of culturemedium. After a 48-hours incubation at 37° C. the cell culture mediumwas replaced by fresh medium in which Extract A according to theinvention was dissolved at different concentrations that killed 50% and30% of cell population. After 24 or 72 hours of treatment cells wereharvested in suspension and counted. 250.000 cells were centrifuged for10 minutes at 1700 rpm at 4° C. The pellet was washed with PBS at 4° C.and incubated with annexin V-FITC and propidium iodide solution for 15minutes at 4° C. in the dark. For each sample, data from approximately10,000 cells were recorded on logarithmic scale. Software incorporatedinto the flow cytometer was used to define precisely the percentage ofcells in apoptotic and/or necrotic pathway, and the normal cells. Eachexperiment was realized two times. The mean of percentage of both theapoptotic and the necrotic way and the standard error of the relatedmean was calculated. Each condition was compared to the control, beingnon-treated cells.

Four-fold increase of apoptotic HCT-15 cells was observed when cellswere treated by Extract A at 0.25 μg/ml for 24 hours as compared to thecontrol cells (FIG. 17). The A172 cell line seemed to be engaged in anapoptosis and necrosis pathway when the cells were treated for 24 hoursby Extract A at 25 μg/ml (FIG. 18). This tendency was maintained after72 hours of treatment. The treatment by Extract A induced the apoptosisof J82 cells in a concentration-dependent manner, which is particularlyclear 72 hours after treatment (FIG. 19). Also an increase in thepercentage of necrotic cells is observed at both concentrations at 72hours. A 2.5 fold increase in the percentage of Hs683 apoptotic cellswas obtained after 24 hours of treatment with Extract A at 0.025 μg/ml(FIG. 20). After 72 hours of treatment the percentage of apoptotic cellsincreased in concentration-dependent manner.

In conclusion, Extract A principally induces an increase in thepercentage of apoptotic cells. An effect on the necrosis pathway wasobtained under certain conditions.

Example 5 Maximum Tolerated Dose of an Extract According to theInvention

In the present example, the MTD index was determined for Extract Aaccording to the invention. The Maximum Tolerated Dose (MTD) of a givendrug is defined as the maximum amount of a drug which can beadministered acutely, i.e. in one intraperitoneal, intravenous,subcutaneous or per os single dose, to healthy animals, i.e. animals notgrafted with tumors.

The experimental conditions to determine the MTD index of Extract A werethe following. The survival times of mice, which are not grafted with atumor, were recorded up to 14 days post-Injection. Six different dosesof Extract A were used for the determination of the MTD index. Thehighest dose administered to tumor-bearing mice was 160 mg/kg. Otherdoses comprised 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg and 80 mg/kg. Eachexperimental group was composed of two mice for the determination of theMTD index. Table 4 below shows the data obtained for the MTD index usingExtract A. TABLE 4 MTD index determination for Extract A according tothe invention Administered doses of extract Day 1 post Day 14 post(mg/kg) administration administration 1 × 5  — — 1 × 10 — — 1 × 20 — — 1× 40 X 1 × 80 XX  1 × 160 XXwhere x means one dead mouse and — means all the animals remained alive

According to the definition given above, the MTD index for Extract A is20 mg/kg for single administration in mice. Thus, the Maximum ToleratedDose (MTD) index of Extract A, referring to the maximum amount ofExtract A, which can be administered acutely to healthy animals, is 20mg/kg extract. This value indicates that Extract A has a broadtherapeutic window.

Example 6 In Vivo Effects of an Extract According to the InventionEvaluated on Three Cancer Models

This example illustrates the in vivo effects of the Extract A accordingto the invention on three different cancer models.

The in vivo effects of Extract A were studied on mice grafted withdifferent types of tumors, including a lymphoma cancer, a melanomacancer and a breast cancer. The in vivo effects were evaluated withthree types of parameters:

-   -   the cumulative toxicity, which is evaluated by recording the        weights of the tumor-bearing mice during treatment    -   the actual anti-tumor effect exerted at tumor growth level,        which is evaluated by measuring tumor size three times a week by        means of a caliper. The actual tumor growth is expressed as an        area (mm²) by multiplying the two largest perpendicular        diameters.    -   the survival gain for the mice treated, which is calculated by        means of the T/C index. This index is the ratio between the        median survival time of the group of treated mice (T) and that        of the control group (C). The extract is considered to be active        if the T/C value is above 130% (P<0.05), very active for a value        higher than 150% (P<0.01) and toxic for a value lower than 70%.        In Vivo Effects of Extract A on a Lymphoma Cancer Model

Extract A was evaluated on the aggressive P388 lymphoma cancer model. Ina first experiment three doses, 10 mg/kg, 5 mg/kg and 2.5 mg/kg werecompared to control. The mice were inoculated subcutaneously with 10⁶P388 cells at day D0 and treated nine times during the three followingweeks at days D5, D7, D9, D12, D14, D16, D19, D21 and D23 post-graft.Each experimental group contained nine mice.

FIG. 21A illustrates that Extract A did not influence the body weight ofthe mice. FIG. 21B shows that Extract A significantly induced a decreasein P388 lymphoma growth. The T/C index values for the 7×10 mg/kgadministration schedule gave a T/C index of 111%, the T/C index valuesfor the 6×5 mg/kg schedule gave a T/C index of 100% and the T/C indexvalues for the 8×2.5 mg/kg schedule gave a T/C index of 121%. Inconclusion, these results indicated that administration of Extract Adecreased the growth of the P388 lymphoma cancer but did notsignificantly prolong the survival of the P388 lymphoma-bearing mice atthe tested concentrations.

In a second set of experiments, the number of administrations wasincreased from nine to sixteen, accompanied by a concomitant decrease inthe dose per single administration. The subcutaneously-administereddoses were 2.5 mg/kg, 1.25 mg/kg and 0.63 mg/kg. Extract A wasadministered daily, five days a week, for five consecutive weeks(5×5=25).

FIG. 22B shows that these active Extract A doses of 0.63 mg/kg and 1.25mg/kg had negligible toxic effects since the P388 lymphoma-bearing micelost no weight during treatment. FIG. 22A shows that Extract A markedlydecreased P388 lymphoma growth at doses of both 0.63 mg/kg and 1.25mg/kg. In addition, the T/C index values for the 15×2.5 mg/kgadministration schedule gave a T/C index of 137%, that the T/C indexvalues for the 15×1.25 mg/kg schedule gave a T/C index of 137% and thatthe T/C index values for the 13×0.63 mg/kg schedule gave a T/C index of116%. Thus, the 15 administrations of 2.5 mg/kg Extract A and the 15administrations of 1.25 mg/kg Extract A significantly increased thesurvival of these P388 lymphoma-bearing mice by 37%. Consequently, thetreatments at doses of 2.5 mg/kg and 1.25 mg/kg with Extract A prolongedthe survival of P388 lymphoma-bearing mice.

In conclusion, Extract A according to the invention exerts significantanti-tumor effects on the aggressive P388 lymphoma cancer model withoutany loss of body weight in the animals concerned. Also, unexpectedly,the Extract A according to the invention exerts higher anti-tumoractivity when assayed chronically at low doses, i.e. around 1.25 to 0.63mg/kg, than at high doses, i.e. around 10 mg/kg to 5 mg/kg.

In Vivo Effects of Extract A on a Melanoma Cancer Model

Extract A was evaluated on the aggressive B16 melanoma cancer model.Extract A was subcutaneously administered to mice at doses of 2.5 mg/kg,1.25 mg/kg and 0.63 mg/kg. Extract A was administered daily, five days aweek, for five consecutive weeks.

FIG. 23A shows that the administered doses had negligible toxic effectssince the B16-melanoma-bearing mice lost no body weight during thetreatment. FIG. 23B shows that Extract A administrations dispensed at2.5 or 1.25 mg/kg significantly decreased the growth of the B16melanoma. In addition, the T/C index values for the 20×2.5 mg/kgadministration schedule gave a TIC index of 117%, the T/C index valuesfor the 20×1.25 mg/kg schedule gave a T/C index of 117% and the T/Cindex values for the 25×0.63 mg/kg schedule gave a T/C index of 140%.Thus, Extract A decreased the growth of the B16 melanoma andsignificantly prolonged the survival of the B16 melanoma-bearing mice,especially when Extract A was administered 25 times at 0.63 mg/kg whenthe level of significance is reached.

In a second set of experiments, Extract A was administered orally (peros).

FIG. 24A shows that the administered doses had negligible toxic effectssince the B16-melanoma-bearing mice lost no body weight during thetreatment. FIG. 24B shows that the Extract A administrations per os at2.5 or 1.25 mg/kg significantly decreased the growth of the B16 melanomaup to 25 days post-graft. In addition, the T/C index values for the19×2.5 mg/kg administration schedule gave a T/C index of 114%, the T/Cindex values for the 15×1.25 mg/kg schedule gave a T/C index of 100% andthe T/C index values for the 16×0.63 mg/kg schedule gave a T/C index of104%. Thus, Extract A induced a decrease in the growth of the B16melanoma tumor and slightly prolonged the survival of the B16melanoma-bearing mice at 2.5 mg/kg.

In conclusion the two sets of experiments provide evidence that ExtractA according to the invention exerts significant anti-tumor effects onthe B16 melanoma model independent of the mode of administration.

In Vivo Effects of Extract A on a Breast Cancer Model

Extract A was evaluated on the MXT-HI breast cancer model, i.e. ahormone insensitive variant of breast cancer. The MXT-HI modelcorresponds to an undifferentiated carcinoma with dramatic metastaticprocesses towards the liver. It therefore corresponds to the lateclinical stages of human breast cancer. The MXT-HI represents a veryaggressive biological tumor model.

The MXT-HI breast cancer is induced in mice by subcutaneously injectingMXT tumor fragments into the flanks of B6D2F1 mice. Without treatment,the inoculated mice die between the fourth and seventh week after theinoculation. Extract A was assayed at 10 mg/kg, 5 mg/kg and 2,5 mg/kgwith nine administrations, i.e. three times a week for three consecutiveweeks.

FIG. 25A indicates that the various Extract A treatment schedules usedin the present experiments induced essentially no major toxic-sideeffects since the MXT-HI-bearing tumor mice did not lose significantweight. FIG. 25B shows that nine administrations of 2.5 mg/kgsignificantly decreased MXT-HI tumor growth. The T/C index values forthe 9×10 mg/kg administration schedule gave a T/C index of 103%, the T/Cindex values for the 9×5 mg/kg schedule gave a T/C index of 103% and theT/C index values for the 9×2.5 mg/kg schedule gave a T/C index of 119%.Thus at the dose of 2.5 mg/kg, Extract A prolonged the survival of theMXT-HI-bearing mice.

In FIG. 26 the number of the MXT-HI breast cancer bearing mice, whichdied during the experiment in the control (untreated) and test groups(treated with extract), is shown. A Kaplan-Meier statistical analysiswas used and represents the death rate of the nine mice in each group.The statistic value underlines the general fit of the test group incomparison with the general fit of the control group with regard tosurvival.

In conclusion the experiments described above provide evidence thatExtract A exerts significant anti-tumor effects on the MXT-HI breastcancer tumor.

Conclusions on Example 6

The Extract A according to the invention has a significant anti-tumoreffect on the tested cancer models. These models represent a broad panelof histological tumor types, including carcinomas, lymphomas andmelanomas. These models are clinically relevant because they mimicspecific clinical stages of human cancers. Apart from this, they arealso biologically aggressive and invasive because two of themmetastasize dramatically to the liver.

While having a very significant anti-tumor effect, Extract A accordingto the invention exhibits negligible toxic effects since thetumor-bearing mice did not lose body weight during the treatments. Thesemi-purified extract can therefore include an “antidote” in addition tothe anti-tumor compound responsible for all the anti-tumor activitiesreported here.

Surprisingly, Extract A induces significantly higher anti-tumoractivities when assayed chronically at low doses i.e. around 1.25 to0.63 mg/kg, than at high doses, i.e. around 10 mg/kg to 5 mg/kg.

Example 7 In Vivo Toxicological Effects of an Extract According to theInvention

This example illustrates the toxicological effects of Extract Aaccording to the invention, in particular the in vivo hematotoxicity andgeneral toxicity.

Hematotoxicity

Hematotoxicity is evaluated by establishing hematological profiles foreach animal species. Particular attention was paid to the numbers ofblood platelets, red cells and leukocytes. The effect of Extract A wasevaluated at two doses, i.e. 5 mg/kg and 1.25 mg/kg, by theintra-peritoneal administration to mice. The administration schedule wasfive administrations a week for five consecutive weeks resulting in atotal injection number of twenty-five. The animals were sacrificed threedays after the last injection. There were ten mice per group.

As compared to control (FIG. 27), no statistically significant changeswere observed with respect to hematological parameters after treatmentwith Extract A. No significant changes were observed on the mean cellvolume of red blood cells, the mean corpuscular hemoglobin, the meancorpuscular hemoglobin concentration and the platelets after thetreatments.

General Toxicity

The general toxicity of Extract A was histologically tested on mice bymeans of conventional histopathological analyses ofhematoxilin-eosin-stained histological slides obtained from differenttypes of organs and tissues. The effect of Extract A was evaluated at 5mg/kg and at 1.25 mg/kg by intra-peritoneal administration. Theadministration schedule was five administrations a week for fiveconsecutive weeks resulting in a total injection number of twenty-five.The animals were sacrificed three days after the last injection. Thebrain, the heart, the liver, the pancreas, the stomach, the intestinesand the ovaries were collected. There were ten mice per group.

Examination of the collected organs showed that control (untreated)group did not show any particular modification in the organs. Also intreated mice the brain, the heart, the liver, the pancreas, the stomach,the intestines and the ovaries were not affected by Extract A treatmentsat doses of 5 mg/kg or 1.25 mg/kg, indicating that Extract A does nothave a general toxic effect.

Example 8 Anti-Poisonous Effects of Extracts According to the Invention

This example illustrates the anti-poisonous effect of the extractsaccording to the invention. Female mice of 4 to 5 weeks of age wereinjected with two or four times the maximal tolerable dose of twowell-known and clinically used anti-tumor drugs (MTDx4) for adriamycineand (MTDx2) for vincristine. The mice were given a singleintraperitoneal injection with 40 mg/kg body weight adriamycine or asingle intraperitoneal injection of 20 mg/kg body weight vincristine atDay 0. Lot 1 in FIG. 28 shows the survival curve of the mice injectedwith either the MTDx2 of vincristine or either the MTDx4 of adriamycine.

Extract A according to the invention was injected intraperitoneally at adose of 10 mg/kg body weight prior to the injection of the anti-tumordrugs. The following different schedules were applied.

-   -   Extract A was injected on the same day (i.e. at Day 0), but 4        hours prior to the injection of the therapeutic compound        adriamycine or vincristine. Results of this treatment are        represented by lot 2, in FIG. 28.    -   Extract A was injected once a day during eight days prior to the        day of injecting the therapeutic compound adriamycine or        vincristine. Results of this treatment are represented by lot 3,        in FIG. 28.    -   Extract A was injected once a day during eight days prior to the        day of injecting the therapeutic compound adriamycine or        vincristine and was further once a day during seven days after        the injection of the anti-tumor drugs. Results of this treatment        are represented by lot 4, in FIG. 28.

As can be seen in FIG. 28, all schedules wherein Extract A is used incombination with a therapeutic compound significantly prolong thesurvival of the mice as compared to injection of the therapeuticcompounds as single agents.

A second example illustrates the anti-poisonous effect of Extract A withanother administration schedule. The mice were given a singleintraperitoneal injection with 20 mg/kg body weight vincristine at Day0. Lot 1 in FIG. 29 shows the survival curve of the mice injected withof vincristine.

Extract A was injected intraperitoneally at a dose of 10 mg/kg bodyweight prior to or in the same time than the injection of anti-tumordrug. The following different schedules were applied.

Extract A was injected on the same day (i.e. at Day 0), but 6 hoursprior to the injection of the therapeutic compound vincristine. Resultsof this treatment are represented by lot 2, in FIG. 29.

-   -   Extract A was injected on the same day (i.e. at Day 0), but 4        hours prior to the injection of the therapeutic compound        vincristine. Results of this treatment are represented by lot 3,        in FIG. 29.    -   Extract A was injected on the same day (i.e. at Day 0), but 2        hours prior to the injection of the therapeutic compound        vincristine. Results of this treatment are represented by lot 4,        in FIG. 29.    -   Extract A was injected on the same day (i.e. at Day 0) and in        the same time than the injection of the therapeutic compound        vincristine. Results of this treatment are represented by lot 5,        in FIG. 29.

The parameter, which was applied for determining the protective, i.e.anti-poisonous effect of Extract A, on the toxic effects induced by theanti-tumor drugs consisted of the “prolongation of survival”.Statistical analysis was performed by means of Kaplan-Meier statistics.The level of significance was p<0.05.

As can be seen in FIG. 29, the schedule wherein Extract A was injected 4hours prior to the injection of vincistine (lot 3) significantlyprolongs the survival of mice as compared to injection of thetherapeutic compound as single agent.

It can thus be concluded that Extract A enables to significantly protectmice from the toxic effects of a mortal dose of frequently usedanti-tumor compound such as adriamycine or vincristine. Extract Aaccording to the invention has thus an anti-poisonous effect, by whichenables to reduce the toxic effects of well-known anti-tumor compound.

A third example illustrates the anti-poisonous effect of Extract BFemale mice of 4 to 5 weeks of age were injected of four well-known andclinically used anti-tumor compound: adriamycine, vincristine,oxaliplatine and camptothecine. The mice were given a singleintraperitoneal injection with 20 mg/kg body weight adriamycine or asingle intraperitoneal injection of 20 mg/kg body weight vincristine ora single intraperitoneal injection of 40 mg/kg body weight oxaliplatineor a single intraperitoneal injection of 20 mg/kg body weightcamptothecine at Day 0.

Lot 1 in FIG. 30, FIG. 31, FIG. 32, FIG. 33 shows the survival curve ofthe mice injected with either adriamycine or either vincristine oreither oxaliplatine or either camptothecine.

Lot 2 in FIGS. 30 to 33 shows the “survival point” of the mice injectedwith Extract B at 10 mg/kg.

Lot 3 in FIGS. 30 to 33 show the survival curve of the mice injectedintraperitoneally with Extract B at 10 mg/kg body weight on the same day(i.e. at Day 0) but 4 hours prior to the injection of the therapeuticcompound.

As can be seen in FIGS. 30 to 33, Extract B used in combination with atherapeutic compound significantly prolongs the survival of the mice ascompared to injection of the therapeutic compound as single agents.Extract B didn't show cytotoxic properties. Indeed, no mouse died whenit was injected with Extract B as single agent. Statistical analysis wasperformed by means of Kaplan-Meier statistics. The level of significancewas p<0.05 for each combination Extract B and therapeutic compound.

It can thus be concluded that Extract B provides significant protectionto mice from the toxic effects of a mortal dose of frequently usedanti-tumor drugs. Extract B has thus an anti-poisonous effect, whichenables the reduction of the toxic effects of well-known anti-tumordrugs. The anti-poisonous activity was preserved in Extract B as inExtract A.

A fourth example illustrates the anti-poisonous effect of Extract C.Extract C was used in combination of adriamycine at 20 mg/kg bodyweight.

Lot 1 in FIG. 34 shows the survival curve of the mice injected with 20mg/kg body weight of adriamycine.

Lot 2 in FIG. 34 show the survival curve of the mice injected withExtract C at 10 mg/kg on the same day (i.e. at Day 0) but 4 hours priorto the injection of the therapeutic compound.

As can be seen in FIG. 34, the injection of Extract C induced asignificant survival prolongation of the mice as compared to theinjection of the therapeutic compound as single agent. It can be thusconcluded that Extract C show an anti-poisonous properties as theprevious Extract B.

A fifth example illustrates the anti-poisonous effect of Extract D.Female mice of 4 to 5 weeks of age were injected with adriamycine. Themice were given a single intraperitoneal injection with 20 mg/kg bodyweight adriamycine at Day 0 (Lot 1 in FIG. 35). Lot 2 in FIG. 35 showsthe survival curve of the mice injected with Extract D intraperitoneallyat a dose of 5 mg/kg body weight on the same day (i.e. at Day 0), but 4hours prior to the injection of the therapeutic compound adriamycine.

As can be seen in FIG. 35, the injection of Extract D induced asignificant survival prolongation of the mice as compared to theinjection of the therapeutic compound as single agent. It can be thusconcluded that Extract D show an anti-poisonous properties as theprevious Extract C.

In conclusion, these experiments illustrate that the purified extractsaccording to the invention prolong significantly the survival of mice ascompared to the mice injected with therapeutic compound as single agent.Extract D, the most purified extract, showed an anti-poisonousproperties on the toxic effects induced by the anti-tumor drugs.

1. A method of treating cancer comprising administering a compositioncomprising an extract of a Calotropis procera plant, and at least onetherapeutic compound and/or a physical treatment that exerts relevant,detrimental side effects on normal, non-cancer related cells, tissues ororgans to an individual in need of treatment.
 2. The method according toclaim 1, wherein said extract is obtained using an extraction procedure,comprising the steps of: a) extracting a starting material of saidCalotropis procera plant, in an aliphatic alcohol, by dissolving thestarting material in said alcohol thereby obtaining a suspension of saidmaterial in said alcohol, stirring said suspension, and filtering saidsuspension by fritted glass thereby obtaining a first filtrate and afirst solid part; b) extracting said first solid part in an aliphaticalcohol thereby obtaining a second filtrate and a second solid part; c)combining said first and said second filtrate thereby obtaining acombined filtrate, and evaporating said combined filtrate under vacuumthereby obtaining said extract, wherein said starting material isselected from the group consisting of fruits, aerial parts, subterraneanparts, and their mixtures.
 3. The method according to claim 1, wheresaid extract comprises at least two active compounds selected from thegroup consisting of asclepin, calactin, vorusharin, calotropin,calotropagenin, uzarigenin, calotoxin, usharin, usharidin, and2″oxo-vorusharin.
 4. The method according to claim 1, wherein the weightratio of extract: therapeutic compound is in the range 0.001:1 to1000:1.
 5. The method according to claim 1, wherein said cancer isselected from the group consisting of breast cancer, lymphoma, sarcoma,pancreatic cancer, melanoma, colorectal cancer, glioma, non small celllung cancer, small cell lung cancer, skin cancer, bone cancer, ovariancancer, CNS cancer, renal cancer, bladder cancer, head and neck cancer,prostate cancer, liver cancer, and hematological cancers.
 6. The methodaccording to claim 1, wherein said therapeutic compound(s) is ananti-cancer agent.
 7. The method according to claim 1, wherein saidtherapeutic compound is selected from the group consisting ofadriamycin, alkeran, ara-c, bleomycin, biCNU, busulfan, CCNU,carboplatinum, cisplatinum, cyclophosphamide, cytoxan, daunorubicin,DTIC, 5-FU, fludarabine, gemcitabine (gemzar), herceptin,hexamethylmelamine, hydrea, idarubicin, ifosfamide, irinotecan(camptosar, CPT-11), leustatin, methotrexate, mithramycin, mitomycin,mitoxantrone, muphoran, navelbine, nitrogen mustard, oxaliplatine,rituxan, STI-571, streptozocine, taxol, taxotere, topotecan (hycamtin),velban, vincristine, VP-16, xeloda (capecitabine), and zevelin.
 8. Themethod according to claim 1, wherein said therapeutic compound(s) is acytotoxic antibody or a fragment thereof.
 9. The method according toclaim 1, wherein said therapeutic compound(s) is a cytotoxic hormone ora fragment thereof.
 10. The method according to claim 1, wherein saidtherapeutic compound(s) is a cytotoxic peptide or a fragment thereof.11. The method according to claim 1, wherein said physical treatment istherapeutic radiation.
 12. The method according to claim 1, wherein saidextract is administered prior to, after, or at the same time as saidtherapeutic compound(s).
 13. A method for obtaining an extract havingbiologically active components comprising the steps of: a) extracting astarting material of a Calotropis procera plant in an aliphatic alcohol,by dissolving the starting material in said alcohol thereby obtaining asuspension of said material in said alcohol, stirring said suspension;and filtering said suspension by fritted glass thereby obtaining a firstfiltrate and a first solid part; b) extracting said first solid part inan aliphatic alcohol thereby obtaining a second filtrate and a secondsolid part; c) combining said first and said second filtrate therebyobtaining a combined filtrate; and d) evaporating said combined filtrateunder vacuum thereby obtaining said extract, wherein said startingmaterial is selected from the group consisting of fruits aerial partssubterranean parts, and their mixtures.
 14. An active extract isolatedfrom the method according to claim
 13. 15. (canceled)
 16. A kitcomprising a container in which an extract of Calotropis procera asdefined in claim 1 is present, and a container in which a therapeuticcompound is present.